There is a need for rapid detection of bacteria and fungi in clinical specimens and there is a requirement to distinguish between a bacterial and fungal infection. Culture approaches can be used but such techniques require several days to complete, especially when attempting to detect small numbers of bacteria and also when detecting slower growing micro organisms.
Tests may be carried out based upon measuring the presence of a molecule which can be linked to the presence in the sample of a bacterial or fungal cell. The most commonly detected molecule is Adenosine Triphosphate (ATP). Detection of DNA and RNA has also been proposed, although the correlation between the presence of DNA and RNA and viability is not clear-cut due to the variable persistence of nucleic acids in specimens post death of the microorganism (Keer & Birch, Journal of Microbiological Methods 53 (2003) 175-183). Detection of adenylate kinase as an indicator of viability has also been proposed (Squirrel) D J, Murphy M J, Leslie R L, Green J C D: A comparison of ATP and adenylate kinase as bacterial cell markers: correlation with agar plate counts. In Bioluminescence and chemiluminescence progress and current applications. Edited by: Stanley R A, Kricka L J. John Wiley and Sons; 2002 and WO 96/02665)
A routinely employed method for determining ATP levels involves the use of bioluminescence. The method uses the ATP dependency of the reaction in which light emitting luciferase catalyzes oxidation of luciferin. The method may be used to measure relatively low concentrations of ATP. Kits useful for detecting ATP using bioluminescence are commercially available from Roche, New Horizons Diagnostics Corp, Celsis etc.
Ligases are enzymes which catalyze ligation of nucleic acid molecules. The ligation reaction requires either ATP or NAD+ as co-factor depending upon the ligase concerned. WO 2009/007719 describes the use of NAD ligases to detect viable bacteria.